1. Field of the Invention
The invention is related to methods and reagents for inhibiting tumor cell growth. Specifically, the invention identifies genes necessary for tumor cell growth as targets for developing drugs to inhibit such genes and thereby inhibit tumor growth. The invention provides methods for screening compounds to identify inhibitors of said genes, and methods for using said inhibitors to inhibit tumor cell growth. The invention also provides peptides encoded by genetic suppressor elements of the invention and mimetics and analogues thereof for inhibiting tumor cell growth. Also provided by the invention are normalized random fragment cDNA libraries prepared from tumor cells of one or a plurality of tumor cell types wherein the cDNA fragments can be induced by treating recipient cells with a physiologically-neutral stimulating agent.
2. Summary of the Related Art
The completion of the draft sequence of the human genome has provided the art with a partial list of known and putative human genes, the total number of which is estimated to be between 30,000 and 45,000 (Venter et al., 2001, Science 291: 1304–1351; Lander et al., 2001, Nature 409: 860–921). These genes provide many potential targets for drugs, some of which may be useful in preventing the growth of cancers. However, the development of clinically useful gene-targeting anticancer drugs could be greatly facilitated by the ability to narrow down the list of human genes to those that are involved in the primary feature of cancer, uncontrolled tumor growth. It would be especially useful to identify genes necessary for the growth of tumor cells and to determine which of the genes play a tumor-specific role and are not required for normal cell growth. These genes are particularly attractive targets for developing tumor-specific anticancer agents.
Most of the effort in tumor-specific drug targeting in the prior art has focused on oncogenes, the function of which has been associated with different forms of cancer Perkins and Stem (1997, in CANCER: PRINCIPLES AND PRACTICE OF ONCOLOGY, DeVita et al., eds., (Philadelphia: Lippincott-Raven), pp. 79–102). Oncogene targets have been viewed in the art as being more “tumor-specific” than “normal” cellular enzymes that are targeted by the drugs used in present chemotherapeutic regimens. The tumor specificity of oncogenes has been suggested primarily by the existence of oncogene-associated genetic changes, such as mutations or rearrangements, specific to neoplastic cells. Although oncogenes are mutated or rearranged in some cases, in other cases they are merely expressed at elevated levels or at inappropriate stages of the cell cycle, without changes in the structure of the gene product (Perkins and Stem, 1997, Id.). Even when mutated, proteins encoded by oncogenes rarely acquire a qualitatively novel function relative to the “normal” protooncogene products. Hence, products of mutated, rearranged or overexpressed oncogenes generally perform the same biochemical functions as their normal cell counterparts, except that the functions of the activated oncogene products are abnormally regulated.
It is noteworthy that none of the “classical” oncogenes known in the art have been identified as targets for clinically useful anticancer drugs discovered by traditional mechanism-independent screening procedures. Rather the known cellular targets of chemotherapeutic drugs, such as dihydrofolate reductase (inhibited by methotrexate and other antifolates), topoisomerase II (“poisoned” by epipodophyllotoxins, anthracyclines or acridine drugs), or microtubules that form the mitotic spindle (the targets of Vinca alkaloids and taxanes) are essential for growth and proliferation of both normal and neoplastic cells. Tumor selectivity of anticancer drugs appears to be based not merely on the fact that their targets function primarily in proliferating cells, but rather on tumor-specific response to the inhibition of anticancer drug targets. For example, Scolnick and Halazonetis (2000, Nature 406 430–435) disclosed that a high fraction of tumor cell lines are deficient in a gene termed CHFR. In the presence of antimicrotubular drugs, CHFR appears to arrest the cell cycle in prophase. CHFR-deficient tumor cells, however, proceed into drug-impacted abnormal metaphase (Scolnick and Halazonetis, 2000, Id.), where they die through mitotic catastrophe or apoptosis (Torres and Horwitz, 1998, Cancer Res. 58: 3620–3626). In addition to CHFR, tumor cells are frequently deficient in various cell cycle checkpoint controls, and exploiting these deficiencies is a major direction in experimental therapeutics (O'Connor, 1997, Cancer Surv. 29: 151–182; Pihan and Doxsey, 1999, Semin. Cancer Biol. 9: 289–302). In most cases, however, the reasons that inhibition of anticancer drug targets selectively induces cell death or permanent growth arrest in tumor cells are unknown. There is therefore need in the art to identify additional molecular targets in tumor cells, inhibition of which would arrest tumor cell growth.
One method known in the art for identifying unknown genes or unknown functions of known genes is genetic suppressor element technology, developed by some of the present inventors (in U.S. Pat. Nos. 5,217,889, 5,665,550, 5,753,432, 5,811,234, 5,866,328, 5,942,389, 6,043,340, 6,060,134, 6,083,745, 6,083,746, 6,197,521, 6,268,134, 6,281,011 and 6,326,488, each of which is incorporated by reference in its entirety). Genetic suppressor elements (GSEs) are biologically active cDNA fragments that interfere with the function of the gene from which they are derived. GSEs may encode antisense RNA molecules that inhibit gene expression or peptides corresponding to functional protein domains, which interfere with protein function as dominant inhibitors. The general strategy for the isolation of biologically active GSEs involves the preparation of an expression library containing randomly fragmented DNA of the target gene or genes. This library is then introduced into recipient cells, followed by selection for the desired phenotype and recovery of biologically active GSEs from the selected cells. By using a single cDNA as the starting material for GSE selection, one can generate specific inhibitors of the target gene and map functional domains in the target protein. By using a mixture of multiple genes or the entire genome as the starting material, GSE selection allows one to identify genes responsible for a specific cellular function, since such genes will give rise to GSEs inhibiting this function. In a variation of this approach, the vector used for library preparation contains sequences permitting regulated expression of cDNA fragments cloned therein.
This method can be used to identify genes required for tumor cell growth by subjecting the cells to negative growth selection. One example of this type of selection is known in the art as bromodeoxyuridine (BrdU) suicide selection, which has long been used to select conditional-lethal mutants (Stetten et al., 1977, Exp. Cell Res. 108: 447–452) and growth-inhibitory DNA sequences (Padmanabhan et al., 1987, Mol. Cell Biol. 7: 1894–1899). The basis of BrdU suicide selection is the destruction of cells that replicate their DNA in the presence of BrdU. BrdU is a photoactive nucleotide that incorporates into DNA and causes lethal DNA crosslinking upon illumination with white light in the presence of Hoechst 33342. The only cells that survive this selection are cells that do not replicate their DNA while BrdU is present, such as cells that express growth-inhibitory genes or GSEs. One advantage of this method is very low background of surviving cells. When used with GSE libraries under the control of an inducible vector, this selection method excludes spontaneously arising BrdU-resistant mutants by the insensitivity of their phenotype to the presence or absence of the inducing agent. Another major advantage of this technique is its sensitivity for weak growth-inhibitory GSEs: even if only a small fraction of GSE-containing cells are growth-inhibited by GSE induction, such cells will survive BrdU suicide and will give rise to a recovering clone.
The applicability of this approach to the isolation of growth-inhibitory GSEs was first demonstrated by Pestov and Lau (1994, Proc. Natl. Acad. Sci. USA 91: 12549–12553). These workers used an IPTG-inducible plasmid expression vector to isolate cytostatic GSEs from a mixture of cDNA fragments from 19 murine genes associated with the G0/G1 transition. In this work, three of the genes in the mixture gave rise to growth-inhibitory GSEs (Pestov and Lau, 1994, Id.). In a subsequent study, Pestov et al. (1998, Oncogene 17: 13187–3197) used the same approach to isolate one full-length and one truncated cDNA clone with growth-inhibitory activity from a 40,000-clone library of nominally full-length mouse cDNA. However, the method disclosed in the art cannot be efficiently used for transducing a library of random fragments representing the total mRNA population from a mammalian cell such as a tumor cell because the method relies on plasmid expression vectors for library construction, and only a limited number of cells can be stably transfected by such libraries.
There remains a need in the art to discover novel genes and novel functions of known genes necessary for tumor cell growth, especially by using methods for identifying genes based on function. There is also a need in the art to identify targets for therapeutic drug treatment, particularly targets for inhibiting tumor cell growth, and to develop compounds that inhibit the identified targets and thereby inhibit tumor cell growth.